CN101539550A - Qualitative and quantitative analysis method for polyoses - Google Patents
Qualitative and quantitative analysis method for polyoses Download PDFInfo
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Abstract
The invention relates to a qualitative and quantitative analysis method for polyoses, which comprises the following steps: (1) the zymohydrolysis and the verification of the polyoses: measuring the content of a polyoses water solution by a colorimetric method; (2) comparing the change of a polyoses mapping before and after zymohydrolysis by HPSEC-ELSD analysis; (3) saccharide ingredient direct HPLC analysis of a polyoses zymohydrolysis solution or HPLC analysis after pre-column derivatization: identifying polyoses mapping and chromatogram peaks by taking a standard monosaccharose, a uronic acid, a disaccharide, and the like as contrasts and taking a chromatogram peak mass-to-charge ratio as reference, and using stable characteristic sections as a quantitative analysis index; and (4) realizing the qualitative and quantitative analysis of the polyoses by establishing polyoses zymohydrolysis responding characteristics and polyoses zymohydrolysis mapping which are based on structural information through the independent or combining application of the steps. The qualitative and quantitative analysis method can be used for the identification and quantitative measurement of the polyoses of traditional Chinese medicine, such as panax, panax pseudoginseng, gen-seng, aweto, cordyceps, ganoderma lucidum, fomes japonica, milk veteh, angelica, and the like and provides an effective method for controlling the quality of the polyoses and the products thereof.
Description
Technical field
The present invention relates to a kind of qualitative and quantitative analytical approach that is used for polysaccharide.
Background technology
Polysaccharide is that a class is anhydrated by 10 above monose condensations, with the poly carbohydrates of glycosidic bond form in conjunction with formation.As requisite composition in all living organisms, it has close getting in touch with the energetic supersession and the multiple physiological function that earn a bare living.In recent years, the polysaccharide in source such as plant, sea life and mushroom is because of its significant biologically active such as antitumor [Lin Meng sense, Deng. Chinese herbal medicine, 2007,38:949-953.], immunological regulation [Fu Shujie, etc. the time precious traditional Chinese medical science traditional Chinese medicines, 2008,19:99-101.], hypoglycemic [Luo Zuyou, Deng. Food Science, 2007,28:596-600.], anti-oxidant [Chen Xuan, Deng. Chinese Journal of New Drugs, 2007,16:1000-1004.] and anti-inflammatory antiviral [the fourth insurance money is etc. Chinese Pharmaceutical Journal, 2004,39:561-563] etc. caused that people pay close attention to widely.Yet because the structure and the composition of polysaccharide complexity, its quality control does not have breakthrough for a long time.The molecular weight of polysaccharide, composition monose, conformation and glycosidic bond link etc. are closely related with its activity, but how to differentiate rapidly and accurately that the polysaccharide of separate sources is still the key issue that solution is needed in polysaccharide and products thereof quality control badly so far.
At present, by structure elucidation carry out method that polysaccharide differentiates loaded down with trivial details time-consuming, equipment requirements is high, difficulty is big, poor practicability.Though use by research and to form the monose feature and ratio is carried out the polysaccharide discriminating, acid-hydrolyzed poor selectivity, the easy destroyed loss of monose under the acid condition and cause distortion as a result.
Summary of the invention
The object of the invention is to provide a kind of polysaccharide fast and accurately qualitative, quantitative analysis method.This method will overcome loaded down with trivial details time-consuming, the deficiencies such as equipment requirements is high, difficulty is big, poor practicability of prior art.Realize easy, practical qualitative, the quantitative test of polysaccharide.
Compare with acid hydrolysis, enzyme hydrolysis has good, the characteristics such as specificity is high, mild condition of selectivity, can obtain the specificity hydrolysate, and specific restriction endonuclease hydrolysis properties can characterize the architectural feature of polysaccharide, thereby helps the identification of polysaccharide.In addition, the peptide spectrum that enzymolysis forms in conjunction with stratographic analysis extensively become the strong instrument that polypeptide and protein differentiates [Yan Yan, etc. analytical chemistry, 2006, it is plain gorgeous that 34:187-190. opens, etc. SCI, 2002,23:1246-1250.].In view of this, the present invention propose using polysaccharides to the response characteristic of different glycoside hydrolases in conjunction with stratographic analysis, make up corresponding " the sugar spectrum " that characterizes polysaccharide structures information, realize polysaccharide and products thereof fast, accurate qualitative identification; And be index with the characteristic fragment, realize the quantitative test of polysaccharide.
The objective of the invention is to be achieved through the following technical solutions: a kind of qualitative and quantitative analytical approach that is used for polysaccharide is characterized in that step is as follows:
(1), the enzymolysis of polysaccharide and checking: polysaccharide solution is with sulfuric acid-phynol colorimetric method for determining content, regulate sugared content, some parts of polysaccharide solutions of parallel preparation, add an amount of glycosidase respectively and carry out enzymolysis, the amount ranges of the employed enzyme of every 1mg (with glucose meter) polysaccharide is 3U～650U, and each enzyme digestion reaction repeats 3～5 times; This step in order to, 1) determine the enzymolysis feature of polysaccharide; 2) reproducibility of affirmation enzymolysis;
(2), HPSEC-ELSD analyzes: polysaccharide solution and zymolyte thereof are analyzed through HPSEC-ELSD, relatively the variation of the forward and backward polysaccharide collection of illustrative plates of enzymolysis; Polysaccharide collection of illustrative plates enzymolysis is forward and backward significant change does not take place to be designated as feminine gender (-) response; Have in the collection of illustrative plates characteristic peak disappearance, reduce, the appearance of perhaps new chromatographic peak then is designated as the positive (+) response;
(3), HPLC analysis behind direct HPLC analysis of carbohydrate content or the pre-column derivatization in the polysaccharide enzymolysis liquid: the oligosaccharides and the monose of residual fragment of polysaccharide enzymolysis and/or generation, available reversed-phase HPLC-ELSD are analyzed and are obtained sugared composing; Also can pass through derivatization, improve separating effect and the ultraviolet absorption characteristic of carbohydrate content on reversed-phase column, improve the sensitivity of ultraviolet detection and Mass Spectrometer Method, and then improve qualitative, quantitative accuracy; Serve as contrast and be reference with standard monose, uronic acid, disaccharide etc., carry out sugar and compose chromatographic peak and discern with chromatographic peak mass-to-charge ratio (m/z); Its stable characteristics fragment is then as the quantitative test index.
(4), independence or use in conjunction by above steps, can make up polysaccharide enzymolysis response characteristic and polysaccharide (enzymolysis) sugar spectrum based on structural information, realize qualitative, quantitative test to polysaccharide.
The qualitative and quantitative analytical approach that is used for polysaccharide of the present invention is the following principle of work of utilization:
1, polysaccharide is to the hydrolysising characteristic of specificity glycosidase: adopt efficient size exclusion chromatograph (HPSEC) analysis, many sugars are not through different glycosidases such as araban restriction endonuclease (endo-arabinanase, EC3.2.1.99), zytase (xylanase, EC 3.2.1.8), 1,4-β-D-galactan restriction endonuclease (endo-1,4-β-D-galactanase, EC 3.2.1.89), cellulase (cellulase orendo-1,4-β-D-glucanase, EC 3.2.1.4), pectase (pectinase orendopolygalacturonase, EC 3.2.1.15), AMS (α-amylase, EC 3.2.1.1), isoamylase (isoamylase, EC 3.2.1.68), 1,3-beta glucan restriction endonuclease (endo-1,3-β-D-glucanase, EC3.2.1.39), laminarinase (lichenase, EC 3.2.1.73), 'beta '-mannase (β-mannanase, EC 3.2.1.78), 1,3 (4)-beta glucan restriction endonuclease (endo-1,3 (4)-β-glucanase, EC 3.2.1.6), hyaluronidase (hyaluronidase, EC 3.2.1.35) and dextranase (dextranase, EC3.2.1.11) etc. before the hydrolysis, back HPSEC chromatogram changes as the disappearance at polysaccharide peak or reduces and the appearance of chromatographic peak (enzymolysis product) newly, and clear and definite polysaccharide is to the hydrolysis response characteristic of different glycosidases;
2, sugared analysis of spectrum: adopt HPLC-ELSD directly to analyze above-mentioned polysaccharase and separate thing, or with above-mentioned zymolyte behind derivatization (as: reduction amination, with pyrazoline ketone, hydrazine class reagent reacting, amino derivatization, hydroxyl derivatization, carboxyl derivatization etc.), analyze with HPLC-DAD-ELSD or HPLC-DAD-MS/MS, obtain polysaccharase hydrolysis products characteristic spectrum-sugar spectrum, with this qualitative identification polysaccharide; Characteristic fragment with the polysaccharide enzymolysis is an index, select as polysaccharide such as standard glucosan (dextran), blue polysaccharide (pullulan) sugar in general Shandong, the cellotriose enzymolysis characteristic fragment of arbitrary compound or purifying to the monose such as disaccharide such as oligosaccharides, maltose, cellobiose, galactobioses such as cellohexose, grape trisaccharide to six sugar or glucose, galactose, mannose is a reference substance, realization is at the quantitative test of polysaccharide structures feature.
Say that more specifically and more optimally step of the present invention is as follows:
Step (1), the enzymolysis of polysaccharide and checking: polysaccharide solution is measured content with the phenolsulfuric acid method, regulate sugared content, some parts of polysaccharide solutions of parallel preparation, add an amount of glycosidase respectively and carry out enzymolysis, enzymatic hydrolysis condition is based on each enzyme service manual recommend method or optimize back use (seeing Table 1), and each enzyme digestion reaction repeats 3～5 times.This step in order to, 1) determine the enzymolysis feature of polysaccharide; 2) reproducibility of affirmation enzymolysis.
The enzymatic hydrolysis condition of table 1 polysaccharide (based on the operation manual of Megazyme and Sigma company carbohydrase)
Step (2), HPSEC-ELSD analyzes: polysaccharide solution and zymolyte thereof are analyzed through HPSEC-ELSD, relatively the variation of the forward and backward polysaccharide collection of illustrative plates of enzymolysis.Polysaccharide collection of illustrative plates enzymolysis is forward and backward significant change does not take place to be designated as feminine gender (-) response; Have in the collection of illustrative plates characteristic peak disappearance, reduce, the appearance of perhaps new chromatographic peak then is designated as the positive (+) response.
Step (3), HPLC analysis behind direct HPLC analysis of carbohydrate content or the pre-column derivatization in the polysaccharide enzymolysis liquid: the oligosaccharides and the monose of residual fragment of polysaccharide enzymolysis and/or generation, available reversed-phase HPLC-ELSD are analyzed and are obtained sugared composing; Also can pass through derivatization, improve separating effect and the ultraviolet absorption characteristic of carbohydrate content on reversed-phase column, improve the sensitivity of ultraviolet detection and Mass Spectrometer Method, and then improve qualitative, quantitative accuracy.Serve as contrast and be reference with standard monose, uronic acid, disaccharide etc., carry out sugar spectrum chromatographic peak and discern with the chromatographic peak mass-to-charge ratio.Its stable characteristics fragment is then as the quantitative test index.
By the independence or the use in conjunction of technique scheme, can make up polysaccharide enzymolysis response characteristic and polysaccharide (enzymolysis) sugar spectrum based on structural information, realize qualitative, quantitative test to polysaccharide.
The qualitative and quantitative analytical approach that is used for polysaccharide of the present invention, can be used for the discriminating of herbal polysaccharides such as genseng, pseudo-ginseng, American Ginseng, Cordyceps sinensis, Cordyceps militaris, red sesame, purple sesame, the Radix Astragali and Radix Angelicae Sinensis and the quantitative measurement of ganoderan, for the quality control of polysaccharide and products thereof provides effective method.
Description of drawings
Fig. 1, Fig. 2 are respectively among the embodiment 21, the HPLC-DAD figure of the product of 4-β-D-galactan enzyme hydrolysis pseudo-ginseng and astragalus polyose behind the PMP derivatization;
Fig. 3, Fig. 4 are respectively among the embodiment 21, the HPLC-MS figure of the product of 4-β-D-galactan enzyme hydrolysis pseudo-ginseng and astragalus polyose behind the PMP derivatization;
Fig. 5, Fig. 6, Fig. 7 are respectively the HPLC-DAD figure of product behind the PMP derivatization of pectase hydrolysis American Ginseng, Cordyceps sinensis and Cordyceps militaris polysaccharide among the embodiment 3;
Fig. 8, Fig. 9, Figure 10 are respectively the HPLC-MS figure of product behind the PMP derivatization of pectase hydrolysis American Ginseng, Cordyceps sinensis and Cordyceps militaris polysaccharide among the embodiment 3;
Figure 11, Figure 12 are respectively the HPLC-ELSD figure that the product of pectase hydrolysis glossy ganoderma and purple sesame polysaccharide among the embodiment 4 is directly measured;
Figure 13, Figure 14 are respectively the HPLC-DAD figure of product behind the PMP derivatization of pectase hydrolysis glossy ganoderma and purple sesame polysaccharide among the embodiment 4;
Figure 15, Figure 16 are respectively the HPLC-ELSD figure of product behind the PMP derivatization of pectase hydrolysis glossy ganoderma and purple sesame polysaccharide among the embodiment 4.
Embodiment
Embodiment 1:
The extraction of herbal polysaccharide: take by weighing genseng, pseudo-ginseng, American Ginseng, Cordyceps sinensis, Cordyceps militaris, red sesame, purple sesame, the Radix Astragali and 9 kinds of each 0.5g of Chinese medicine medicinal powder of Radix Angelicae Sinensis, the 100 ℃ of high-temperature water refluxing extraction of water 1 hour that add 20 times of w/vs, extract obtains supernatant through centrifuging, getting the 5mL supernatant, to add absolute ethyl alcohol to final concentration be 75%, 4 ℃ of following standing over night precipitations, centrifuged deposit is through 4mL 95% ethanol washed twice, evaporate to dryness alcohol in 60 ℃ of water-baths, residue redissolves with 5mL hot water (60 ℃), and the centrifuging and taking supernatant gets polysaccharide extraction liquid.With the phenolsulfuric acid colourimetry is quantitative reference, and regulating extract is that 0.15mg/mL (with glucose meter) is standby to containing sugared relative content.
The enzymolysis of polysaccharide: get many parts of 1mL extracts and add the araban restriction endonuclease respectively, zytase, 1,4-β-D-Galactanase, cellulase, pectase, AMS, isoamylase, 'beta '-mannase, 1,3-beta glucan restriction endonuclease, laminarinase, 1,3 (4)-beta glucan restriction endonucleases, hyaluronidase, dextranase to final concentration is 1.4U/mL, 4.5U/mL, 9.4U/mL, 10U/mL, 95U/mL, 12.5U/mL, 10U/mL, 0.5U/mL, 4.6U/mL, 10U/mL, 5U/mL, 5U/mL, 5U/mL, mixed liquor acts on spend the night (〉=12 hours) by the hydrolysising condition of recommending (seeing Table 1).Hydrolyzate was ended enzymolysis process in 30 minutes with 85 ℃ of heating.Centrifugal back supernatant is cooked HPSEC respectively and is analyzed.
HPSEC-ELSD analyzes: Agilent 1100 series of high efficiency liquid chromatographs are equipped with quaternary pump, automatic sample handling system and Agilent chromatographic work station.Chromatographic column is TSK G-4000PW
XLGel column (300mm * 7.8mm i.d., 10 μ m), 30 ℃ of column temperatures; Elution requirement is a 20mmol/L ammonium acetate aqueous solution isocratic elution, flow velocity 0.6mL/min; 110 ℃ of ELSD2000ES drift tube temperatures, nitrogen carrier gas flow velocity 3.0L/min, ram cuts out; Sampling volume 10 μ L.
Testing result, with reference to table 2: carry polysaccharide based on each water and can distinguish panaxan and Radix Angelicae Sinensis polysaccharide to 13 kinds of hydrolysis response characteristics that glycosidase showed, and other three groups: pseudo-ginseng and astragalus polyose, American Ginseng, Chinese caterpillar fungus and Cordyceps militaris polysaccharide, red sesame and purple sesame polysaccharide.
Liquid medicine was carried polysaccharide during the response of table 2. enzymolysis was differentiated 9 kinds
Embodiment 2:
The extraction of herbal polysaccharide: take by weighing pseudo-ginseng and each 0.5g of Milkvetch Root powder, all the other operations are with the extraction step of polysaccharide among the embodiment 1.
The enzymolysis of polysaccharide: with embodiment 1.
HPSEC-ELSD analyzes: with embodiment 1.
The PMP derivatization of polysaccharide enzymolysis liquid: get supernatant 600 μ L after the herbal polysaccharide enzymolysis liquid is centrifugal, mix with equal-volume ammoniacal liquor, add the methanol solution 200 μ L of 0.5mol/LPMP again, the mixing sealing is reacted 30min down in 70 ℃.Taking-up is put and is chilled to room temperature, shakes up after adding 2mL water, with 50 ℃ of following evaporates to dryness, twice of repetitive operation.Residue adds the water-soluble back of separating of 1mL with the equal-volume chloroform extraction, and three times repeatedly, water layer is analyzed for HPLC-DAD-MS after crossing 0.45 μ m filter membrane.
HPLC-DAD-MS analyzes: the serial LC/MSD of Agilent 1200 system is equipped with binary pump, automatic sample handling system, Agilent chromatographic work station.Chromatographic column is Zorbax Eclipse XDB-C
18, 25 ℃ of column temperatures; Moving phase is 20mmol/L ammonium acetate aqueous solution (A) and acetonitrile (B), gradient elution, and program is 0～1min, 13～15%B, 1～40min, 16%B; Secondary array pipe detector, wavelength 245nm; Mass detector, electric spray ion source, the positive ion detecting pattern, the ion full scan is by m/z 100 to 1500, nitrogen drying gas velocity 8L/min, 350 ℃ of baking temperatures, atomizing pressure 45psi, second order ms separates width 4, fragment amplification 1.5, compound degree of stability 50%; Flow velocity 1.0mL/min; Sampling volume 10 μ L.
Testing result, with reference to table 2, Fig. 1 to Fig. 4: it is approximate to 13 kinds of hydrolysis response characteristics that glycosidase showed that pseudo-ginseng and Radix Astragali water are carried polysaccharide.Both enzymolysis products, with 1,4-β-D-Galactanase enzymolysis is an example, HPLC-DAD and HPLC-MS analyze and all can obtain characteristic spectrum and be used for differentiating behind the PMP derivatization.
Embodiment 3:
The extraction of herbal polysaccharide: take by weighing American Ginseng, Chinese caterpillar fungus and each 0.5g of Cordyceps militaris medicinal powder, all the other operations are with the extraction step of polysaccharide among the embodiment 1.
The enzymolysis of polysaccharide: with embodiment 1.
HPSEC-ELSD analyzes: with embodiment 1.
The PMP derivatization of polysaccharide enzymolysis liquid: with embodiment 2.
HPLC-DAD-MS analyzes: with embodiment 2.
Testing result, with reference to table 2, Fig. 5 to Figure 10: it is approximate to 13 kinds of hydrolysis response characteristics that glycosidase showed that American Ginseng, Cordyceps sinensis and Cordyceps militaris water are carried polysaccharide.Three's enzymolysis product is an example with the pectinase enzymatic hydrolysis, and HPLC-DAD and HPLC-MS analyze and all can obtain characteristic spectrum and be used for differentiating behind the PMP derivatization.
Embodiment 4:
The extraction of herbal polysaccharide: take by weighing red sesame and purple each 0.5g of sesame medicinal powder, all the other operations are with the extraction step of polysaccharide among the embodiment 1.
The enzymolysis of polysaccharide: with embodiment 1.
HPSEC-ELSD analyzes: with embodiment 1.
HPLC-ELSD analyzes: Agilent 1100 series of high efficiency liquid chromatographs are equipped with quaternary pump, automatic sample handling system and Agilent chromatographic work station.Chromatographic column is Prevail
TMCarbohydrate ES chromatographic column (250mm * 4.6mm i.d., 5 μ m), 25 ℃ of column temperatures, moving phase is water (A) and acetonitrile (B), 75% isocratic elution, flow velocity 1.0mL/min; 110 ℃ of ELSD2000ES drift tube temperatures, nitrogen carrier gas flow velocity 3.0L/min, ram cuts out; Sampling volume 10 μ L.
The PMP derivatization of polysaccharide enzymolysis liquid: with embodiment 3.Preparation liquid is analyzed for HPLC-DAD-ELSD.
HPLC-DAD-ELSD analyzes: Agilent 1100 series of high efficiency liquid chromatographs are equipped with quaternary pump, automatic sample handling system and Agilent chromatographic work station.Chromatographic column is Zorbax EclipseXDB-C
18, 25 ℃ of column temperatures; Moving phase is 20mmol/L ammonium acetate aqueous solution (A) and acetonitrile (B), gradient elution, and program is 0～1min, 13～15%B, 1～40min, 16%B; Secondary array pipe detector, wavelength 245nm; 110 ℃ of ELSD2000ES drift tube temperatures, nitrogen carrier gas flow velocity 3.0L/min, ram cuts out; Sampling volume 10 μ L.
Testing result, with reference to table 2, Figure 11 to Figure 16: it is approximate to 13 kinds of hydrolysis response characteristics that glycosidase showed that red sesame and Zi Zhishui carry polysaccharide; But both pectase hydrolysates, directly HPLC-ELSD measures, or HPLC-DAD-ELSD measures behind the PMP derivatization, all can obtain characteristic spectrum and be used for differentiating.
Embodiment 5:
The extraction of herbal polysaccharide: take by weighing red sesame medicinal powder 0.5g, all the other operations are with the extraction step of polysaccharide among the embodiment 1.
The enzymolysis of polysaccharide: with the pectase is hydrolytic enzyme, and all the other are operated with enzymolysis step among the embodiment 1.
HPLC-ELSD analyzes: with embodiment 4.
Quantitative analysis results, with reference to Figure 11 and Figure 12: with the A characteristic peak is index, is contrast with the standard glucose, can calculate the amount that can be accounted for crude drug by the feature polysaccharide of pectin enzyme spcificity hydrolysis is 0.38mg/g.
Claims (5)
1, a kind of qualitative and quantitative analytical approach that is used for polysaccharide is characterized in that step is as follows:
(1), the enzymolysis and the checking of polysaccharide: polysaccharide solution is regulated sugared content with colorimetric method for determining content, and some parts of polysaccharide solutions of parallel preparation add an amount of glycosidase respectively and carry out enzymolysis, and each enzyme digestion reaction repeats 3～5 times;
(2), polysaccharide enzymolysis analysis: polysaccharide solution and zymolyte thereof are analyzed through HPSEC-ELSD, relatively the variation of the forward and backward polysaccharide collection of illustrative plates of enzymolysis; Polysaccharide collection of illustrative plates enzymolysis is forward and backward significant change does not take place to be designated as feminine gender (-) response; Have in the collection of illustrative plates characteristic peak disappearance, reduce, the appearance of perhaps new chromatographic peak then is designated as the positive (+) response;
(3), sugared analysis of spectrum: the oligosaccharides and the monose of residual fragment of enzymolysis and/or generation in the polysaccharide enzymolysis liquid, directly analyze with reversed-phase HPLC-ELSD and to obtain the sugar spectrum; Or, improve separating effect and the ultraviolet absorption characteristic of carbohydrate content on reversed-phase column by pre-column derivatization, improve the sensitivity of ultraviolet detection and Mass Spectrometer Method, and then improve qualitative, quantitative accuracy; Serve as contrast and be reference with standard monose, uronic acid, disaccharide etc., carry out sugar spectrum chromatographic peak and discern with the chromatographic peak mass-to-charge ratio; Its stable characteristics fragment is then as the quantitative test index;
(4), independence or use in conjunction by above steps, make up polysaccharide enzymolysis response characteristic and polysaccharide enzymolysis sugar spectrum based on structural information, realize qualitative, quantitative test to polysaccharide.
2, the qualitative and quantitative analytical approach that is used for polysaccharide according to claim 1, it is characterized in that, are meant " with colorimetric method for determining content " described in the step (1) and " add an amount of glycosidase and carry out enzymolysis ": content is measured in carbohydrate chrominance responses such as phenolsulfuric acid method or anthrone-sulfuric acid process, and use comprises the araban restriction endonuclease, zytase, 1,4-β-D-galactan restriction endonuclease, cellulase, pectase, AMS, isoamylase, 1,3-beta glucan restriction endonuclease, laminarinase, 'beta '-mannase, 1,3 (4)-beta glucan restriction endonucleases, any two or more combinations are carried out enzymolysis to polysaccharide in the glycosidase such as hyaluronidase and dextranase, and the amount ranges of the employed enzyme of every 1mg polysaccharide is 3U～650U.
3, the qualitative and quantitative analytical approach that is used for polysaccharide according to claim 1, it is characterized in that, sugared analysis of spectrum described in the step (3) is meant: adopt HPLC-ELSD directly to analyze above-mentioned polysaccharase and separate thing, or with above-mentioned zymolyte behind derivatization, analyze with HPLC-DAD-ELSD or HPLC-DAD-MS/MS, obtain polysaccharase hydrolysis products characteristic spectrum-sugar spectrum, with this qualitative identification polysaccharide; Characteristic fragment with the polysaccharide enzymolysis is an index, and selecting suitable polysaccharide or oligosaccharides is reference substance, realizes the quantitative test at the polysaccharide structures feature.
4, the qualitative and quantitative analytical approach that is used for polysaccharide according to claim 3, it is characterized in that the derivatization of the zymolyte described in the step (3) is meant: reduction amination, with pyrazoline ketone, hydrazine class reagent reacting, amino derivatization, hydroxyl derivatization, carboxyl derivatization.
5, the qualitative and quantitative analytical approach that is used for polysaccharide according to claim 3, it is characterized in that, described " selecting suitable polysaccharide or oligosaccharides ": be to be selected from: the blue polysaccharide of standard glucosan, general Shandong, cellotriose are to oligosaccharides, maltose, cellobiose, the galactobiose of cellohexose, grape trisaccharide to six sugar, or the enzymolysis characteristic fragment of arbitrary compound or purifying is a reference substance in the monose such as glucose, galactose, mannose, realizes the quantitative test at the polysaccharide structures feature.
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